Assays to determine the concentration of analyte in clinical, environmental, or other settings generally involve the use of serial dilution. The purpose of such dilution is twofold: it may be necessary to bring the concentration of the sample within the range of the assay; if the sample is too concentrated, a meaningful reading may not result. Additionally, serial dilution may accommodate and span a dynamic range wherein variable readings over a series of concentrations is obtained, thus enhancing the precision of the result. In other cases, the level of dilution itself may be used as an assessment of concentration. In this instance, immunoassays or other specific binding assays can be used to assess the quantity of an analyte in a sample by using a multiplicity of test regions or portions in combination with serial dilutions of the sample. A variety of test formats is used wherein the same test format is used in these multiple test regions, but the sample containing analyte is used in lower and lower concentrations until a discernible response disappears. By taking account of the level of dilution at which a response is no longer visible, and comparing the results to those obtained with standards, the concentration of analyte in the original sample can be back-calculated.
Methods that employ serial dilution are useful, but quite labor- or machine-intensive, and are not suited for semiquantitative determinations as might be needed in testing for analytes outside of a laboratory context. For example, ascertaining the levels of contaminants in soil at the location where field testing is appropriate should be accomplished by methods that require only the application of a single sample volume, rather than the more complex and error-prone process of obtaining multiple dilutions. Similarly, in clinical settings, shortages of trained and reliable personnel manually to conduct serial dilutions for assessment is a recognized problem in supplying health care; instrumentation to make such dilutions mechanically is expensive and of limited reliability. Furthermore, it would be desirable to conduct clinical assays on extremely small samples so as to minimize the invasive nature of sample taking. Conduct of serial dilution on samples in the microliter range, for example, is inherently inaccurate.
One approach to this problem has been set forth in U.S. Pat. Nos. 4,654,310 and 4,923,800 to Ly. In the methods described, systematically varying amounts of test reagents in multiple test portions are used to obtain semiquantitative results for the same solution of analyte without necessity for serial dilutions of the sample. One easily understood disclosed approach takes advantage of two competing catalytically controlled reactions using varying relative amounts of the two catalysts. In its simplest form, two enzymes which utilize the analyte as the substrate compete for conversion of the substrate to product. One of the products gives an all-or-none detectable result; the other does not give a detectable response. If there is a high concentration of analyte, even large amounts of competing enzyme which take away from conversion to the detectable product don't matter; however, at low concentrations of analyte, not enough will be left to see the result. Therefore, high concentrations of analyte will be capable of giving a detectable result in the presence even of high concentrations of the competing enzyme; low concentrations of analyte will only give a detectable result at low concentrations of competing enzyme. In a somewhat different, but related, approach, described in U.S. Pat. 4,042,329 to Hochstrasser, variable stoichiometric reagent concentrations are used to achieve semiquantitative results in a series of test regions.
The present invention similarly provides a method that permits quantitative analyte concentration determination using a series of test regions without the need for serial dilution. In contrast to the abovementioned techniques, the invention method takes advantage of the variable binding affinity of a multiplicity of ligands either with the analyte itself or with a specific binding partner of the analyte. In either case, the invention takes advantage of a multiplicity of ligands which react with varying degrees of efficacy for a single substance.